Assessment of optimized FRET substrates as universal corona- and picornavirus main protease substrates for screening assays

Abstract

Coronaviral infections are an important cause of enteric and respiratory diseases in humans and animals that are generally associated with a high level of morbidity and mortality. Similarly, picornavirus infections can lead to various illnesses that severely impact human and animal health. Despite belonging to different virus families, viral replication in all of these pathogens relies on the action of a central cysteine protease called 3C/3CL or main protease (M^pro). Due to the high functional and structural conservation of this enzyme among viral species and robustness against mutation it is considered a good target for antiviral inhibitor development. The evaluation of inhibitor potency, expressed as IC₅₀, in many studies is achieved by measuring the inhibition of cleavage of a fluorogenic substrate in a Fluorescence Resonance Energy Transfer (FRET)-type assay. The FRET substrate is engineered after common recognition sequences of each viral M^pro, resulting in different sequences and limited comparability of IC₅₀ between species. Our aim was to overcome this inconsistency by identifying common recognition motives of coronavirus and picornavirus M^pros to develop a unique FRET substrate that can be used universally for FRET assay tests of these enzymes. We synthesized a variety of FRET substrates with common recognition sequences and compared their cleavage kinetics towards main proteases from different species to determine the optimal sequence for universal application in FRET assays.